CD22 and Siglec-G are B lymphocyte specific glycan binding proteins that participate in regulation of B cell receptor signaling. The extracellular domain recognizes sialic acid containing glycans as ligands, which are abundantly expressed as glycan chains all mammalian cells. In this project we have sought to understand how CD22 regulates the activation of B lymphocytes and, in particular, to understand the impact of glycoprotein ligands on CD22 function. Over the last decade, the major focus has been on the roles of cis ligands on the B cell, which mask the ligand-binding site of CD22. However, we now recognize that trans ligands on opposing cells have profound impact on CD22 in the context of B cell receptor recognition of antigens on the same cell. During the past project period we have obtained evidence that a major role of trans ligands of B cell siglecs, CD22 and Siglec-G/10, is to enforce tolerance to membrane antigens in the periphery. Tolerance induction involves recruitment of B cell siglecs to an immunological synapse between the B cell and an opposing cell containing both the antigen and siglec ligands, comprising sialic acid containing glycans present on all mammalian cells. The enforced juxtaposition of siglecs with the B cell receptor (BCR) triggers a siglec-mediated tolerogenic circuit that results in apoptosis of the antigen reactive B cell. Data in support of this mechanism have been obtained using nanoparticle platforms developed for multivalent display of both antigen and synthetic siglec ligands. We now have evidence that antigens displayed on cell membranes with natural sialoside ligands also invoke robust tolerance in a siglec dependent manner. Based on these and related findings, the aims of the next project period are directed towards documenting roles of siglecs and their ligands in B cell tolerance, and defining the major signaling components of the tolerogenic circuit that mediates apoptosis of antigen reactive B cells. In particular, we will: 1) Investigate the roles of CD22 and Siglec-G and their natural ligands in peripheral tolerance; 2) Assess the contributions of signaling components that define a tolerogenic circuit induced by B cell siglecs; and 3) Assess the potential for Siglec-G to contribute to central B cell tolerance. These aims will be pursued using a variety of chemical and genetic tools and approaches that have been developed in the past project period.